Faculty of Science
DO CONDITIONS ON THE MEXICAN MOULTING GROUNDS INFLUENCE
CAROTENOID CONTENT AND COMPOSITION OF BULLOCK’S ORIOLE (ICTERUS
BULLOCKII) FEATHERS?
2016 | KAITLIN L. SPARROW

B.Sc. Honours thesis

DO CONDITIONS ON THE MEXICAN MOULTING GROUNDS INFLUENCE
CAROTENOID CONTENT AND COMPOSITION OF BULLOCK’S ORIOLE (ICTERUS
BULLOCKII) FEATHERS?
by
KAITLIN L. SPARROW

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENTS OF BIOLOGICAL AND
PHYSICAL SCIENCES
(Chemical Biology)

This thesis has been accepted as conforming to the required standards by:

Matthew Reudink (Ph.D.), Thesis Supervisor, Dept. Biological Sciences
____________________________________________________
Kingsley Donkor (Ph.D.), Co-supervisor, Dept. Physical Sciences
____________________________________________________
Nancy Flood (Ph.D.), Examining Committee member, Dept. Biological Sciences
____________________________________________________

© Kaitlin L. Sparrow, 2016

ABSTRACT
The Bullock’s oriole (Icterus bullockii) has an interesting moult migration strategy, in
which it stops mid-way to the wintering grounds, in the Mexican monsoon region to replace
feathers (moult) before continuing migration south to overwinter. Moult-migrants, such as
Bullock’s orioles, undergo such a strategy because food resources are more abundant in the
monsoon region during early fall than on the breeding grounds, allowing better conditions for
moult. During this time, Bullock’s orioles produce bright orange/yellow carotenoid-based
plumage. Carotenoid colouration is an honest signal of individual quality and is important in
social interactions and female mate choice across taxa. Here, we asked whether diet and habitat
quality during moult (inferred by stable isotopes) were associated with feather carotenoid content
and composition. Liquid chromatography mass spectrometry (LC-MS) was used to determine
carotenoid concentrations in the breast feathers of after second year males of a Bullock’s oriole
population breeding in British Columbia. Feathers were also analyzed for carbon (δ13C) and
nitrogen (δ15N) stable isotopes to infer habitat conditions during moult. We also used reflectance
spectrometry to calculate feather hue, red chroma, and brightness. Perhaps surprisingly, I found
no direct relationship between features of colouration (as measured by reflectance spectrometry)
and carotenoid concentration or composition. There was, however, a positive relationship
between δ15N and concentration of lutein, as well as a negative relationship between δ15N and
proportion of canthaxanthin. These results suggest that feeding at a higher trophic level during
moult –perhaps having a diet high in insects –allows for a greater amount of lutein to be
deposited in feathers. Feeding more on insects may also indicate a lack of β-carotene or dietary
canthaxanthin, or an inability to effectively metabolize β-carotene to canthaxanthin in that
environment, possibly due to stress. This study highlights the importance of moulting in highquality habitats for species that exhibit carotenoid-based colouration. A moult migration strategy
may have evolved to take advantage of superior moult conditions in the Mexican monsoon
region, which can directly influence the ability to obtain bright colouration, critical for inter- and
intra-sexual interactions.
Thesis Supervisor: Associate Professor Matthew Reudink

ii

ACKNOWLEDGEMENTS
This research would not be possible without the help and supervision of Dr. Matthew
Reudink and Dr. Kingsley Donkor. Both were extremely passionate, patient, and encouraging
throughout. I am extremely thankful for all the time and knowledge they have given me. Thanks
to the Departments of Chemistry and Biology for resources, Mr. Dave Pouw for maintaining the
LC/MS, Western Economic Diversification Canada for providing funds to purchase the LC/MS,
and Garrett Whitworth for extra help with the LC/MS. Funding for this project was provided by
an NSERC USRA and Dr. Reudink’s NSERC grant.

iii

TABLE OF CONTENTS
ABSTRACT ........................................................................................................................................... i
ACKNOWLEDGEMENTS................................................................................................................ iii
INTRODUCTION................................................................................................................................ 1
MATERIALS AND METHODS......................................................................................................... 6

Fieldwork ....................................................................................................................................................... 6
Stable Isotope Analyses .............................................................................................................................. 6
Colour Analysis ............................................................................................................................................ 7
Standards ....................................................................................................................................................... 7
Extractions..................................................................................................................................................... 7
LC-MS ............................................................................................................................................................ 8
Statistical Analysis ....................................................................................................................................... 9

RESULTS .......................................................................................................................................... 10
Carotenoid concentration and composition in adult male breast feathers ................................. 10
Plumage colour and feather carotenoid concentration and composition .................................... 10
Feather isotopes (δ13C, δ15N) and carotenoid concentration and composition........................... 11

DISCUSSION .................................................................................................................................... 13
LITERATURE CITED ..................................................................................................................... 16
APPENDIX........................................................................................................................................ 21

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INTRODUCTION
Sexual selection is a strong evolutionary force that can lead to the elaboration of
ornamental traits. Individuals with larger, more elaborate, and/or more colourful
ornamental traits tend to be favoured by sexual selection, as these traits may be honest
indicators of individual condition or quality, which function in intersexual competition
and/or mate choice. Ornamental plumage colouration, the bright and vibrant feathers
exhibited by many birds, has long fascinated evolutionary biologists (Hill and McGraw
2006). There are various mechanisms that produce colourful plumage, such as deposition
of pigments, including melanin, porphyrins, and carotenoids in feathers, and light
refraction due to feather structure (Hill and McGraw 2006).
Carotenoids are bright red, yellow, and orange organic pigments that have many
functions in animals, including producing colour, boosting immune response, and acting
as antioxidants (Navara and Hill 2003; García-de Blas et al. 2014). Because animals
cannot synthesize carotenoids hey must be obtained through the diet (Völker 1938). In
birds, carotenoids are ingested, and in some cases, subsequently modified into different
carotenoids, and used in the skin, bill, and feathers to produce colouration. To produce
feather colouration, carotenoids can be deposited directly from the diet as yellow dietary
carotenoids, or they can be modified prior to deposition in feathers. Lutein, zeaxanthin, βcarotene, and β-cryptoxanthin, the main dietary carotenoids, can be modified to the
yellow carotenoids, 3’-dehydrolutein, canary xanthophyll, and canary xanthophyll B, or
modified to the red keto-carotenoids such as α-doradexanthin, astaxanthin, canthaxanthin,
and adonirubin (Hill and McGraw 2006; Appendix A). The main modifications to dietary
carotenoids include oxidation of hydroxyl groups to ketones, which produces modified
yellow carotenoids, or the addition of ketones via oxidation, producing red ketocarotenoids. These metabolic conversions appear to be condition dependent, and indicate
that orange/red feathers are most costly to produce (Hill and Johnson 2012).
Because carotenoids are also necessary for many other functions, including
immune processes, vision, growth, lipogenesis, glycolysis, and energy homeostasis,
becoming colourful involves trade-offs among these shared pathways (Hill and Johnson

1

2012). Individuals using large quantities of carotenoids for colouration must be in good
health to do so. For example, Baeta et al. (2008) examined trade-offs between load of an
intestinal parasite (Isospora), and bill colouration in the blackbird (Turdus merula)
Carotenoid supplemented male blackbirds had bills that were more brightly coloured
bills, as well as lower parasite loads. Infected birds without supplementation had reduced
bill colouration, while supplemented infected birds maintained their bill colouration.
Restriction of carotenoids in the diet of male house finches (Haemorhous mexicanus)
resulted in decreased hue, saturation, and brightness of carotenoid-based plumage (Hill
2000). Interestingly, excessively high levels of carotenoids in American goldfinches
(Carduelis tristis) resulted in tissue damage (Huggins et al., 2010).
While individual condition is essential for the expression of optimal colouration,
individuals may also be constrained by habitat quality during moult. Arriero et al. (2006)
examined how carotenoid-based plumage colouration varied across habitats in Eurasian
blue tit (Parus caeruleus) nestlings and found that tits situated in young and structurally
simple forests showed reduced plumage colouration. Nestlings with more intense yellow
plumage were also of a larger body size and had a stronger immune response upon
injection of phytohemagglutinin (PHA, binds to T-cells and stimulates metabolic
activity). Eeva et al. (2009) analyzed plumage colour in great tit nestlings in polluted and
unpolluted areas. In unpolluted areas, higher levels of plumage carotenoid chroma values
and higher plasma carotenoid levels were observed compared to the polluted area.
Nestlings supplemented with carotenoid rich diets grew better (in terms of body mass,
wing length, plasma lutein, and colouration) in the polluted area only. Similarly, great tits
(Parus major) raised in a deciduous forest were more yellow than those raised in a
coniferous forest due to the increased availability of larvae in the former habitat
(Slagsvold and Lifjeld 1985). Ferns and Hinsley (2008) examined blue tits and great tits
(Cyanistes caeruleus) in low and high quality habitats (small and large woods) and
analyzed yellow ventral plumage variation with respect to moult and/or diet. Blue tits
consumed 47% more and great tits 81% more caterpillar flesh in high quality habitats
compared to lower quality habitats. Surprisingly, unlike the results found by Arriero
(2006), this did not result in a significant difference in the chroma of feathers produced
by birds living in the two habitats. In house finches, those occupying environments with

2

high carotenoid availability have larger red patches than those living in dry, hot
environments (Hill et al., 2002). Habitat plays an important role in colouration both
directly, as moulting in a poor-quality habitat can result in an insufficient amount of
carotenoids being consumed and deposited, and indirectly, through malnourishment,
exposure to parasites, high stress load, or other factors, ultimately resulting in a need to
utilize carotenoids elsewhere.
Migratory birds vary vastly in the timing and location of their moult. Which
feathers are replaced and when can vary greatly among species and even within species
across populations. In the arid west, several species hereafter referred to as moultmigrants interrupt their migration to moult in the Mexican monsoon region, located in
southwestern USA and northwestern Mexico (Leu and Thompson, 2002; Rohwer et al.,
2005; Pyle et al., 2009; Pillar et al., 2015). However, this is a vast region that
encompasses a range of habitats; individuals may be able to optimize their colouration by
selecting a high quality moult location. The challenge, however, is tracking habitat use
across seasons in animals moving hundreds or thousands of kilometres between breeding,
moulting, and wintering areas.
Because habitat can influence food and carotenoid availability, moving to areas
with high food availability during moult could provide a strong selective advantage.
Although small migratory birds are difficult to track due to their small sizes and the vast
distances they travel, stable isotope analysis has become quite useful for linking different
phases of the annual cycle and determining diet and habitat use. This technique provides
information on prior location and habitat use (Wassenaar 2008), as stable isotopes are
incorporated into a growing animal’s tissue from the food and water it ingests. Because
stable isotopes in feathers are inert once the feathers are grown, analyzing stable isotopes
of carbon and nitrogen in feathers can provide information on diet and habitat use at the
time of moult.
Stable isotope analysis makes use of the fact that a handful of elements have
forms that vary in mass –referred to as isotopes –that can be distinguished from each
other on analysis. Analysis of carbon (δ13C) and nitrogen (δ15N) stable isotopes in an
animal’s tissues can provide information about the individual’s diet at the time during
that tissue was grown. Carbon (δ13C) stable isotope values can indicate habitat types due

3

to differences in plant water stress and photosynthetic systems (Lajtha and Marshall
1994). Specifically, low δ13C values indicate a diet containing high amounts of C3 plants
(or prey that feeds on C3 plants) and plants experiencing little water stress, while high
δ13C values indicate diets with more C4 plants (Lajtha and Marshall 1994). Differing δ13C
values are also indicative of marine versus terrestrial dietary input (Lajtha and Marshall
1994). Nitrogen (δ15N) stable isotope analysis is associated with the trophic level of an
organism. Organisms that are more carnivorous have a greater δ15N values (Post 2002,
Poupin et al., 2011). Hydrogen (δ2H) stable isotope analysis can provide rough latitudinal
approximation (Hobson 2008). Moving from southeast to northwest North America, δ2H
values become more negative (Hobson 2008). Stable isotope analysis of bird tissues has
been used to establish migratory connectivity between breeding and wintering
populations (Hobson et al., 2004), determine quality of winter habitat (Marra et al. 1998),
and in turn, discern carry-over effects (Marra et al. 1998; Reudink et al., 2009a): events
that occur during one season that influence the performance of individuals the following
season. Recently, hydrogen isotope analysis was used to confirm that a northern
population of Bullock’s orioles (Icterus bullockii) moults between their breeding and
wintering grounds, likely in the Mexican monsoon region (Pillar et al., 2015).
While Pillar et al. (2015) demonstrated that Bullock’s orioles moult-migrate, it
remains unclear how specific habitat and moult conditions affect an individual’s ability to
obtain carotenoids for plumage colouration. Here, we use carbon and nitrogen stable
isotope analysis to ask whether conditions during moult influence carotenoid content and
composition in Bullock’s orioles. Bullock’s orioles are large songbirds that breed as far
north as the southern interior of British Columbia, before overwintering as far south as
Costa Rica. However, these birds stop and fully moult in the Mexican monsoon region
prior to reaching their wintering sites (Rohwer et al., 2005; Pillar et al., 2015). After
second year (ASY) males exhibit bright orange plumage on their breast, belly, rump, and
face, while females of all ages have a pale yellow head and breast. Second year (SY)
males look similar to females, but also exhibit patches of ASY-like plumage. In this
species, older males, who are more orange than younger males, secure higher
reproductive success (Richardson and Burke 1999; Whittingham and Dunn 2000).

4

I predict that Bullock’s orioles that moulted in high-quality, wet habitats in the
Mexican monsoon region (indicated by low δ13C values) will have more carotenoids
deposited in their feathers than those that moulted in low-quality, xeric habitats (high
δ13C values). In addition, I predict that high concentrations of carotenoids, especially
keto-carotenoids such as canthaxanthin, should produce feathers with orange-shifted hue,
high red chroma, and low brightness. However, the relationship between carotenoid
concentration and δ15N may be either positive or negative, depending on the richness of
carotenoids in insects and plants during moult.

5

MATERIALS AND METHODS

Fieldwork
Fieldwork was conducted in Kamloops, British Columbia, Canada (50.68 N
120.34 W). Adult male Bullock’s orioles were captured in mistnets with the use of
conspecific playback and decoys or by positioning nets near oriole feeders. Breast
feathers (n = 10 feathers per bird) were collected from each individual during the summer
(May through July) of 2012 (n = 18) and 2013 (n = 10). Identification of the correct sex
and age class was made using following descriptions provided by Rohwer and Manning
(1990) and Pyle (1997). Individuals were banded with a single Canadian Wildlife Service
aluminum band and three unique colour bands for individual identification.

Stable Isotope Analyses
For all feathers, analysis of stable-carbon and stable-nitrogen isotope ratios was
performed at the Smithsonian Institution Isotope Mass Spectrometry Lab in Suitland,
Maryland, USA. Feathers and claws were washed in a 2:1 chloroform-methanol solution
and allowed to dry. Approximately 0.30-0.40 mg of the distal tip of the feather was
sampled, excluding the rachis. Using a Thermo high temperature conversion elemental
analyzer (TC/EA; Thermo Scientific) at 1350 °C, samples were first pyrolyzed and
sequentially analyzed by a Thermo Delta V Advantage isotope ratio mass spectrometer.
All isotope ratios are reported in δ notation in units per mil (%) relative to international
standards. For carbon stable isotope analysis, following Reudink et al. (2009c), each claw
sample was weighed and converted to CO2 (in an oxidation/reduction furnace) before
separation and quantification of δ13C by gas chromatography equipped with an isotoperatio mass spectrometer (Lajtha and Marshall 1994; Norris et al. 2004). For each stable
carbon and nitrogen analysis, between every 10 samples, two house standards were run
(acetanalide and urea). Repeatability of carbon and nitrogen samples was found to be ±
0.2% (based on repeatability of measurements of standards; Reudink et al., 2015).
Nitrogen (δ15N) was used because 15N is preferentially incorporated with increasing
trophic level, resulting in greater δ15N values at higher trophic levels (Post 2002, Poupin

6

et al., 2011). Furthermore, δ15N values are negatively correlated with rainfall and
positively associated with temperature, so that δ15N level may be indicative of the aridity
of a biome (Sealy et al., 1987; Craine et al., 2009; Reudink et al., 2015).

Colour Analysis
Following Reudink et al. (2009b), reflectance spectrometry was performed on
feathers using an Ocean Optics JAZ spectrometer attached to a PX-2 xenon pulsed light
source to record reflectance spectra across the bird visual spectrum (300-700 nm) from
which variables of hue, red chroma, and brightness were calculated. Feathers were
mounted on a minimally reflective black background (<5% reflectance) and the probe
was kept at a 90° angle. Ten measurements were taken in the centre of the orange region
on the stack of 10 breast feathers for each bird. In between measurements for each bird,
readings from dark (sealed black velvet lined box) and white (spectralon) standards were
used for standardization. Hue, which describes the colour (i.e., yellow, orange, red), was
calculated as arctan ([(R415-510 – R320-415)/R320-700]/[(R575-700 –R415-575)/R320-700]). Red
chroma, which describes the degree of colour saturation, was calculated as the amount of
red light reflected relative to the overall reflectance: (R575-700)/(R300-700). Brightness was
calculated as the mean amount of light reflected across all wavelengths: (mean R300-700).

Standards
Carotenoid standards and vials were purchased from Sigma-Aldrich, Oakville,
ON, Canada, and VWR, Mississauga, ON, Canada, respectively. Standards were made by
weighing out canthaxanthin, lutein, or xanthophyll into a volumetric flask and filling with
a chloroform (CHCl3) and methanol (MeOH) solution (1:1, v/v). Standards were serial
diluted, ranging from 3 to 100 ppm.

Extractions
Feathers were blotched with hexane and allowed to dry. Once dry, feathers were
cut and weighed into glass vials. Anti-bumping granules were added along with acidic

7

pyridine (3 drops of HCl in 50 mL pyridine). The mixture was refluxed for 3 hours. Once
cooled, the carotenoids were transferred to hexane by adding hexane and mixing, then
washing three times with water. The hexane layer was transferred to a new vial and the
drying agent sodium sulfate was added. The extraction was evaporated to dryness with
nitrogen gas, and then a known amount of CHCl3 and MeOH solution (1:1, v/v) was
added. The antioxidant butylated hydroxytoulene (BHT) was also added to prevent
oxidation of the carotenoids (2 μL of 10 mg/mL BHT per sample). The extractions were
kept in glass vials and stored in a dark freezer until use.

LC-MS
Analysis was performed on an Agilent 1200 series HPLC system (Agilent
Technologies, Mississauga, ON, Canada) coupled to an Agilent 6530 Accurate-Mass
Quadrupole Time-of-Flight (Q-TOF) spectrometer, equipped with electrospray ionization
(ESI) source (gas temperature, 300 °C; drying gas, 8 L/min; nebulizer 35 psig; sheath gas
temperature, 350 °C; sheath gas flow, 11 L/min; Vcap, 3500 V). Carotenoids were
analyzed in positive ion mode, and mass spectra were collected between 100 and 600
m/z. A sample of 5 μL of blanks, extractions, and standards (3 to 100 ppm) were injected
into the LC, with the flow rate set at 1.0 mL/min. Separation was achieved with a Luna
C8 (2) column (100 mm × 4.6 mm; 3 μm particle size; Agilent, Canada) kept at a
constant temperature of 60 ± 0.2 °C. The auto-sampler and column were set for 60 °C.
Mobile phase (A) consisted of pure methanol and mobile phase (B) was composed of
70:30 v/v methanol with 0.1% ammonium acetate. Gradient elution was programed as
follows: 95% B at time zero, 80% B at 10 min, 65% B at 15 min, 40% B at 20 min, 10%
B at 24 min, and 95% B again at 25 min, with the effluent flowing into the Q-TOF MS.
Data was obtained by taking the area divided by the retention time of the peak of
interest. Using the data produced from the standards, a calibration curve was produced
for each canthaxanthin, lutein, and xanthophyll. The calibration curves were used to
determine the given carotenoid content in the extraction sample, which in turn was used
to determine the amount of carotenoid in μg per mg of feather extracted. We examined
the repeatability of our analysis by using linear regression on repeated samples. For

8

repeatability varied among carotenoids for both concentration (r2 = 0.30 – 0.53, n = 7)
and proportion of each carotenoid (r2 = 0.19 – 0.31, n = 6).

Statistical Analysis
Canthaxanthin, lutein, and xanthophyll concentrations, as well as total carotenoid
content, were natural log transformed due to strong deviations from normality. I used
linear regression to examine whether hue, red chroma, and brightness were predicted by
carotenoid concentration or by the proportion of each specific carotenoid relative to the
total concentration of all three carotenoids. To examine whether carbon or nitrogen stable
isotope values predicted carotenoid content or concentration, I constructed linear mixed
models with carotenoid content or the proportion of carotenoid present as the response
variable, δ13C and δ15N as fixed effects, and individual as a random effect (due to
repeated measurements of three individuals in different years). Year was also included as
an effect; however, it was not significant in any test and was subsequently removed. All
statistical analyses were performed using JMP v12.0 (SAS 2012).

9

RESULTS

Carotenoid concentration and composition in adult male breast feathers
Concentrations of canthaxanthin, lutein, and xanthophyll ranged from 0.19 to 3.32
μg/mg, 0.42 to 11.4 μg/mg, and 0.20 to 9.88 μg/mg, with means and standard deviations
of 0.59 ± 0.62 μg/mg, 3.75 ± 3.08 μg/mg, and 2.23 ± 1.99 μg/mg, respectively (Fig. 1a).

Figure 1. Distribution of carotenoids in adult male Bullock’s oriole breast feathers. A)
The concentration of carotenoids in μg per mg of feather extracted. B) Proportion
(carotenoid concentration divided by total carotenoid concentration) of each carotenoid.
Boxes show the interquartile range, representing the middle 50% of the data, while
whiskers represent the upper and lower 25% of the distribution; outliers are represented
by stars.

Plumage colour and feather carotenoid concentration and composition
Carotenoid content and composition (canthaxanthin, lutein, and xanthophyll
concentration, total concentration, and proportion canthaxanthin, lutein, and xanthophyll,
Fig. 1b) of feathers did not predict any feather colour variable. (brightness, red chroma,
or hue; all p > 0.12; Table 1).

10

Table 1. Relationships between plumage colour and carotenoid concentration and
composition.
Brightness
Red chroma
Hue
2
2
r = 0.10
r = 0.02
r2 = 0.003
Total
p = 0.12
p = 0.55
p = 0.80
2
2
r = 0.07
r = 0.07
r2 = 0.03
Canthaxanthin
p = 0.20
p = 0.20
p = 0.45
2
2
r = 0.05
r = 0.009
r2 = 0.004
Lutein
p = 0.28
p = 0.66
p = 0.78
Xanthophyll
% Canthaxanthin
% Lutein
% Xanthophyll

r2 = 0.13
p = 0.07
r2 = 0.004
p = 0.77
r2 = 0.02
p = 0.55
r2 = 0.03
p = 0.43

r2 = 0.004
p = 0.76
r2 = 0.04
p = 0.32
r2 = 0.0003
p = 0.94
r2 = 0.003
p = 0.79

r2 = 0.005
p = 0.73
r2 = 0.06
p = 0.25
r2 = 0.002
p = 0.83
r2 = 0.001
p = 0.87

Feather isotopes (δ13C, δ15N) and carotenoid concentration and composition
δ13C was not associated with carotenoid concentration or percentage composition
of carotenoids deposited in feathers (all p > 0.07; Table 2). However, δ15N was correlated
positively with lutein concentration (p = 0.04) and negatively with the proportion of
canthaxanthin in feathers (p = 0.02; Table 1, Fig. 2).

11

Table 2. Relationships between feather isotopes (δ13C, δ15N) and carotenoid
concentration and composition.
δ13C
δ15N
F1, 7.325 = 0.68
F1, 9.589 = 3.73
Total
p = 0.43
p = 0.43
F1, 16.65 = 2.33
F1, 18.59 = 0.02
Canthaxanthin
p = 0.15
p = 0.89
F1, 16.36 = 0.33
F1, 18.84 = 4.79
Lutein
p = 0.58
p = 0.04
F1, 4.935 = 1.19
F1, 6.428 = 1.02
Xanthophyll
p = 0.32
p = 0.35
F1, 20.99 = 3.65
F1, 20.83 = 6.70
% Canthaxanthin
p = 0.07
p = 0.02
F1, 18.42 = 0.06
F1, 20.12 = 2.31
% Lutein
p = 0.81
p = 0.14
F1, 20.56 = 0.28
F1, 20.99 = 0.34
% Xanthophyll
p = 0.60
p = 0.57

Figure 2. Positive relationship between δ15N and lutein concentration, and negative
relationship between δ15N and the proportion of canthaxanthin in Bullock’s oriole breast
feathers.

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DISCUSSION
To our knowledge, this paper is the first to demonstrate that conditions
experienced during moult in the Mexican Monsoon region, as inferred by feather stable
isotopes, are directly associated with carotenoid content and composition. Carotenoids
are important for immune function as well as colouration, providing an honest signal to
both competitors and potential mates (Navara and Hill 2003; Hill and Johnson 2012;
García-de Blas et al. 2014), and are important for signaling across a diversity of animal
taxa (Alfonso et al., 2013; Sefc et al., 2014). Highly ornamented individuals are generally
socially dominant, secure more mates (both social and extra-pair), and attract older
females with greater reproductive capacity (Whittingham and Dunn 2000), and are more
likely to secure paternity at their own nest (e.g., house finch, McGraw and Hill 2000;
American redstart, Reudink et al., 2009b). Because of the importance of obtaining highquality sexual ornaments, individuals should seek out the highest-quality, most nutrient
abundant environment in which to moult in order to achieve vibrant plumage colouration.
Thus, while a moult-migration strategy may have evolved to compensate for low postbreeding food availability in the arid West (Rohwer et al., 2005), habitat selection on the
individual level may be critical for obtaining high-quality plumage, which can then carryover to influence individual success in subsequent seasons.
Carry-over effects (events occurring during one season that affect the
performance in a following season) can drive differences in fitness through effects on
survival and reproduction at both the population and individual level (Harrison et al.,
2011). For example, in the American redstart (Setophaga ruticilla), acquisition of a highquality winter habitats results in the earlier arrival to breeding grounds, and consequently,
positively influences reproductive success (Marra et al., 1998; Reudink et al., 2009b;
Reudink et al., 2009c). Similarly, non-breeding habitat can influence migratory timing in
black-throated blue warblers (Setophaga caerulescens) (Bearhop et al., 2004) and
reproductive success in black-tailed godwits (Limosa limosa islandica; Gunnarsson et al.,
2005). Critically, yellow warblers (Setophaga petechia) undergo a pre-alternate moult of
body feathers on the wintering grounds and Jones et al. (2014) demonstrated that birds
overwintering in higher-quality habitat (inferred via stable isotope analysis) produced
more colourful feathers (higher chroma), which are important for mate choice during

13

breeding. Thus, in moult-migrant birds, this strategy may have evolved to ensure that
birds can acquire carotenoids and nutrients necessary for producing colourful plumage,
thus carrying-over to influence reproductive success.
Recent work by Friedman et al. (2014) on the New World orioles (Icterus) found
that species characterized as yellow had no keto-carotenoids present, while species
exhibiting orange had keto-carotenoids present. When looking across both yellow and
orange species, keto-carotenoid concentration predicted colouration (p < 0.05), but
variation in hue among “orange” taxa was not explained by variation in keto-carotenoid
concentration. Thus, the presence of keto-carotenoids differentiated yellow and orange
colouration, but the concentration of keto-carotenoids within orange taxa did not explain
variation in hue, similar to the lack of relationship we observed between carotenoid
concentration and hue. However, in other species, pigment concentration can accurately
reflect variation in feather hue, as has been demonstrated in the American goldfinch
(Spinus tristis), the house finch (Haemorhous mexicanus), and the Baltimore oriole
(Icterus galbula; McGraw and Gregory 2004; McGraw et al., 2006; Hudon et al., 2013).
Saks et al. (2003) demonstrated that 32-51% of variation in chroma and hue in yellow
areas of greenfinch (Chloris chloris) feathers was explained by carotenoid content.
Bullock’s orioles typically feed on insects, nectar, and fruit during both the winter
and breeding seasons (Rising & Williams 1999). Although diet during moult has not been
studied, the time of moult corresponds to the monsoon season in northwestern Mexico
and southwestern USA, when food is abundant due to the large increase in precipitation
(Rohwer and Manning 1990; Rohwer et al., 2005). Increasing δ15N values, indicating a
higher trophic level during moult, were positively associated with lutein concentration
deposited in Bullock’s oriole feathers. Lutein is one of the main dietary carotenoids and
does not need to be metabolically converted after digestion prior to being deposited into
feathers. Thus, one possibility is that individuals eating a greater quantity of luteincontaining insects may have been able to deposit a greater amount of lutein into their
feathers. Alternatively, individuals eating greater numbers of insects may have been in
better physiological condition and better able to utilize carotenoids for colouration.
Additionally, individuals with lower δ15N values, indicating a lower trophic level
during moult, deposited a greater proportion of their total carotenoid as canthaxanthin.

14

This may be due to feeding on canthaxanthin or β-carotene rich fruit during moult (βcarotene can be metabolically converted into canthaxanthin), which would increase the
amount of canthaxanthin deposited into feathers, but would result in lower δ15N values.
Another possible explanation is that feeding on more insects or canthaxanthin- or βcarotene-poor fruit during moult (perhaps due to poor environmental conditions) may
result in a lack of dietary canthaxanthin or its precursors, resulting in a decreased
proportion of canthaxanthin deposited into feathers. Finally, having a greater proportion
of insects in the diet may be a potential indicator of stress, as this would restrain the
individual to effectively metabolize β-carotene to canthaxanthin for deposition into
feathers (Hill and Johnson 2012), or allocating those carotenoids for use elsewhere (e.g.,
immune function).
Carotenoid concentration repeatability on the LC-MS was relatively low (r2 =
0.30 – 0.53), which may have affected the results; however, our sample size for
repeatability analysis was only seven samples. More samples should be run to examine
the true repeatability of this technique and its utility for carotenoid analysis.
Carotenoid plumage colouration is an important condition-dependent trait that
functions in inter- and intra-sexual communication and can be strongly influenced by
conditions at the time of moult. Here, we demonstrate that plumage colouration in
Bullock’s orioles is directly associated with conditions experienced during moult
stopover in the Mexican monsoon region. We suggest that habitat selection and moult
conditions may carry-over to influence sexual selection and mating dynamics in the
subsequent season. Further, our results are consistent with the idea that a moult migration
strategy may have evolved because both food and carotenoids are more readily available
during the post-breeding period in the Mexican monsoon region than in the arid West.

15

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APPENDIX
Appendix A Modifications of dietary carotenoids (LaFountain et al. 2013). A) β-carotene
to canthaxanthin, via the intermediate, echinenone; B) zeaxanthin to astaxanthin, via the
intermediate, adonixanthin; C) β-cryptoxanthin to adonirubin, via the intermediate, 3hydroxy-echinenone.

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